U.S. patent number 5,214,136 [Application Number 07/482,941] was granted by the patent office on 1993-05-25 for anthraquinone-derivatives oligonucleotides.
This patent grant is currently assigned to Gilead Sciences, Inc.. Invention is credited to Kuei-Ying Lin, Mark Matteucci.
United States Patent |
5,214,136 |
Lin , et al. |
May 25, 1993 |
Anthraquinone-derivatives oligonucleotides
Abstract
Oligonucleotide sequences modified by conjugation to at least
one unsubstituted or substituted anthraquinone at other than the 5'
terminus have favorable properties in enhancing hybridization to
target DNA or RNA without loss of specificity and show enhanced
stability to nucleases.
Inventors: |
Lin; Kuei-Ying (Fremont,
CA), Matteucci; Mark (Burlingame, CA) |
Assignee: |
Gilead Sciences, Inc. (Foster
City, CA)
|
Family
ID: |
23918017 |
Appl.
No.: |
07/482,941 |
Filed: |
February 20, 1990 |
Current U.S.
Class: |
514/44A;
536/24.5 |
Current CPC
Class: |
A61K
31/70 (20130101); C07H 21/00 (20130101) |
Current International
Class: |
C07H
21/00 (20060101); C07H 017/00 (); A61K
031/70 () |
Field of
Search: |
;536/27 ;514/44 |
Other References
Chem. Abst. 114: 78183d, 1991. .
Chem. Abst. 114: 43432g, 1991. .
Ogilvie et al., Pure and Appl. Chem. (1987) 59(3):325-330. .
Froehler et al., Nucleic Acids Res. (1986) 14(13):5399-5407. .
Asseline et al., Tetrahedron Letters (1989) 30(19): 2521-2524.
.
Bayard et al., Biochemistry (1986) 25:3730-3736. .
Lemaitre et al., Nucleosides and Nucleotides (1987) 6
(1&2):311-315. .
Zuckerman et al., Nucleic Acids Res. (1987) 15(13):5305-5321. .
Lancelot et al., Biochemistry (1985) 24:2521-2529. .
Asseline et al., Proc. Natl. Acad. Sci. (1984) 81:3297-3301. .
Mori et al., FEBS Letters (1989) 249(2):213-218. .
van der Krol et al., Biotechniques (1988) 6(10):958-976. .
Stein et al., Cancer Res. (1988) 48:2659-2668..
|
Primary Examiner: Rollins; John W.
Attorney, Agent or Firm: Morrison & Foerster
Claims
We claim:
1. A modified oligonucleotide wherein said modification comprises
at least one substituted or unsubstituted anthraquinone residue
conjugated to a pseudonucleoside.
2. The oligonucleotide of claim 1 wherein said conjugation is
through the .beta.-position of the anthraquinone residue.
3. The oligonucleotide of claim 1 wherein the anthraquinone residue
is unsubstituted.
4. The oligonucleotide of claim 1 wherein said modification
comprises at least two anthraquinone residues.
5. A pharmaceutical composition for treating diseases or conditions
characterized by the presence of unwanted DNA or RNA which
contains, as active ingredient, an effective amount of the
oligonucleotide of claim 1.
Description
TECHNICAL FIELD
The invention relates to oligonucleotides in forms which have been
modified so as to enhance their hybridization ability and stability
with respect to nucleases without decreasing their specificity for
target complementary RNA or DNA. More precisely, the invention
concerns oligonucleotides coupled to at least one anthraquinone
residue.
BACKGROUND ART
The oligonucleotides of the invention are intended, in general, for
application to an approach which has come to be known as
"antisense" therapy.
The general principles of antisense therapy are now well
recognized. Most diseases and undesirable conditions in humans and
animal subjects are mediated by specific DNA or RNA sequences
which, if inactivated, would no longer be able to facilitate the
progress of the disease. The antisense approach provides DNA or RNA
oligomers, or their analogs, which are capable of specific binding
to the undesirable nucleic acid sequences. These materials can be
supplied directly or generated in situ, and may be conventional
oligomers, or are more commonly oligomers having properties which
make them, for example, resistant to nucleases or more capable of
hybridization to the desired target. The hybridization may be
effected by providing oligomers having sequences which result in
conventional base-pairing, or these may recognize double-stranded
DNA by binding to the major or minor grooves which are present in
the double helix. Whatever the ultimate strategy, it is desirable
to provide oligomers with physiological properties which render
them more effective.
The art provides a number of approaches whereby modified
oligonucleotides are used in antisense applications. For example,
in order to provide enhanced stability in vivo, through resistance
to endogenous nucleases, oligomers have been synthesized with
alternative linkages other than the conventional phosphodiester
linkage. Among these are the methylphosphonates wherein one of the
phosphorous-linked oxygens has been replaced by methyl;
phosphorothioates, wherein sulfur replaces one of the oxygens; and
various amidates, wherein NH.sub.2 or organic amine derivatives,
such as morpholidates or piperazidates, replace an oxygen. Also
carbonate and carbamate linkages have been employed, as well as
those involving sulfur rather than oxygen as a linking
substituent.
In addition, modifications have been employed wherein the
oligonucleotides are conjugated with a lipophilic group to enhance
cell permeation capability. Inclusion of intercalators and
chelators which enhance the ability of the oligonucleotide to bind
the target DNA or RNA is also known. These substituents have been
attached to the 5' end of preconstructed oligonucleotides using
amidite or H-phosphonate chemistry, as described by Ogilvie, K. K.,
et al., Pure and Appl Chem (1987) 59:325, and by Froehler, B. C.,
Nucleic Acids Res (1986) 14:5399. Intercalators have also been
attached to the 3' end of oligomers, as described by Asseline, U.,
et al., Tet Lett (1989) 30:2521. This last method utilizes
2,2'-dithioethanol attached to a solid support to displace
diisopropylamine from a 3' phosphonate bearing the acridine moiety
and is subsequently deleted after oxidation of the phosphorus.
Other substituents have been bound to the 3' end of oligomers by
alternate methods, including polylysine (Bayard, B., et al.,
Biochemistry (1986) 25:3730; Lemaitre, M., et al., Nucleosides and
Nucleotides (1987) 6:311) and, in addition, disulfides have been
used to attach various groups to the 3' terminus, as described by
Zuckerman, R., et al., Nucleic Acids Res (1987) 15:5305. It is
known that oligonucleotides which are substituted at the 3' end
show increased stability and increased resistance to degradation by
exonucleases (Lancelot, G., et al., Biochemistry (1985) 24:2521;
Asseline, U., et al., Proc Natl Acad Sci USA (1984) 81:3297). A
recent report by Mori, K., et al., FEBS Lett (1989) 249:213-218,
describes oligonucleotides coupled to anthraquinone at the 5'
terminus. The coupled oligomers exhibit anti HIV activity in vitro;
and the inclusion of the anthraquinone residue appears to raise the
melting temperature. The advantage of this 5' derivatization is
said to be the activity of the anthraquinone as an oxidizing agent
and to produce radicals.
The general approach to constructing various oligomers useful in
antisense therapy has been reviewed by vander Krol, A.R., et al.,
Biotechniques (1988) 6:958-976, and by Stein, C. A., et al., Cancer
Res (1988) 48:2659-2668, both incorporated herein by reference.
The present invention provides oligonucleotides which are coupled
to at least one anthraquinone moiety at other than the 5' terminus;
the inclusion of this moiety is capable of enhancing the ability of
the oligomer to hybridize specifically to target sequences without
loss of specificity. In addition, the anthraquinone can serve as a
marker and can stabilize the oligomer with respect to nuclease
degradation.
DISCLOSURE OF THE INVENTION
The invention is directed to oligonucleotides derivatized to
anthraquinone which can be employed in therapy, for example through
antisense or other mechanisms, or which can be used in a diagnostic
method which involves binding to specific target
oligonucleotides.
Thus, in one aspect, the invention is directed to oligonucleotides
coupled to at least one anthraquinone at other than the 5' terminus
which anthraquinone is unsubstituted or which optionally is
substituted with one or more substituents which do not interfere
with the coupling of the anthraquinone to the oligonucleotide or
with its stability-conferring properties. Examples of such
substituents include alkyl (1-6C)-substituted alkyl with
conventional functional groups such as hydroxyl, carboxyl or the
esters amides or salts thereof; amino groups; sulfhydryl groups;
and halo substituents, such as iodo, bromo, chloro, or fluoro. The
substituents may be in any position of the ring, and may be single
or multiple. The nature of the substituents is limited only by the
availability of the compounds and their noninterference with the
basic properties conferred by the anthraquinone nucleus on the
oligonucleotide to which it is conjugated. The conjugation to the
oligonucleotide is preferably through a beta position of the
anthraquinone and may incorporate a linker moiety between the
anthraquinone and the oligomer.
In other aspects, the invention is directed to pharmaceutical
compositions containing the anthraquinone-derivatized oligomers of
the invention and to methods to bind target DNA using these
compounds and compositions.
MODES OF CARRYING OUT THE INVENTION
Definitions
As used herein, "antisense" therapy refers to administration or in
situ generation of DNA or RNA oligomers or their derivatives which
bind specifically to a target nucleic acid sequence. The binding
may be by conventional base pair complementarity, or, for example,
in the case of binding to DNA duplexes, through specific
interactions in the major groove of the double helix. In general,
"antisense" refers to the range of techniques generally employed
under this description in the art, and includes any therapy which
relies on specific binding to oligonucleotide sequences.
"Oligomers" or "oligonucleotides" includes sequences of more than
one nucleotide and specifically includes short sequences such as
dimers and trimers.
The oligonucleotides in which the pseudo-nucleotides are included
may be conventional DNA or RNA moieties, or may be "modified"
oligomers which are those conventionally recognized in the art. For
example, any of the hydroxyl groups ordinarily present may be
replaced by phosphonate groups, phosphate groups, protected by
standard protecting group, or activated to prepare additional
linkages to additional nucleotides, or may be conjugated to solid
supports. The 5' terminal OH is conventionally phosphorylated; any
2'-OH or OH substituents at the 3' terminus may also be
phosphorylated. The hydroxyls may also be derivatized to standard
protecting groups. The phosphodiester linkage shown may be replaced
by alternative linking groups. These alternative linking groups
include, but are not limited to embodiments wherein P(O)O is
replaced by P(O)S, P(O)NR.sub.2, P(O)R, P(O)OR', CO, or CNR.sub.2,
wherein R is H or alkyl (1-6C) and R, is alkyl (1-6C); in addition,
this group may be attached to adjacent nucleotide through O or S.
Not all linkages in the same oligomer need to be identical.
"Analogous" forms of purines and pyrimidines are those generally
known in the art, many of which are used as chemotherapeutic
agents. An exemplary but not exhaustive list includes
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil,
5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),
wybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
While all of the oligomers of the invention are coupled to at least
one anthraquinone, it is not excluded to include in the
oligonucleotide sequence additional substituents conjugated using
known techniques, such as those coupled through any available OH or
SH moiety, for example, at the 5' position of RNA or DNA, the 2'
position of RNA, or an OH or SH engineered into the 5' position of
pyrimidines.
The oligomers of the invention can be synthesized using standard
solid phase synthesis techniques, for example, using H-phosphonate
chemistry, as described by Froehler, B., et al., Nucleic Acids Res
(1986) 14:5399, or by the methods of Matteucci, M., et al., J Am
Chem Soc (1981) 103:3185. In these approaches, the growing
nucleotide chain is attached to solid support such as
controlled-pore glass (CPG) and extended from the 3' terminus one
nucleotide at a time using a nucleotide protected in the 5'
position; followed by deprotection and addition of a subsequent
nucleotide residue.
In one important approach, the anthraquinone moieties can be
coupled to the oligomer of interest using initial conjugation to a
pseudonucleoside or pseudonucleotide, as described in copending
application U.S. Ser. No. 07/482,943, filed on even date herewith
and incorporated herein by reference. Briefly, these
pseudonucleosides/pseudonucleotides are achiral or are isolated
enantiomers of functional group-bearing organic backbones which can
be incorporated using standard techniques into the synthesis of
oligonucleotides. Other methods of coupling anthraquinone to
preformed oligomers are also known in the art. For example, Nelson,
P. S., et al., Nucleic Acids Res (1989) 17:7179-7186; ibid.,
7187-7194, summarize techniques which are available for
incorporating functionalities into oligonucleotides in a manner
that permits subsequent conjugation to other moieties such as
anthraquinone. Other techniques for conjugating anthraquinone to
oligonucleotide backbones include those set forth in the Background
section above.
The anthraquinone substituent has the general formula ##STR1## with
alpha (1) and beta (2) positions noted as shown. As described
above, the anthraquinone nucleus can be unsubstituted or can be
substituted with one or more substituents which do not interfere
with the properties conferred on the resulting oligomer by the
anthraquinone nucleus. These substituents include alkyl
substituents of limited number of carbons, such as 1-6C; alkynyl
substituents of similar size; and these substituents optionally
substituted with noninterfering functional groups. Halogens or
amino substituents may also be directly substituted onto the
ring.
The anthraquinone substituent is either directly bound to a
functional group present on the oligonucleotide, or can be
conjugated through a linker. For example, linkers which can be
utilized to bind the electrophilic center at the beta position of
the anthraquinone nucleus to additional functional groups on the
oligomer are available from Pierce Chemical Company (Rockford,
Ill.).
Any convenient method of attaching the desired anthraquinone
moieties is within the scope of the invention. The oligomers of the
invention contain at least one anthraquinone moiety at other than
the 5' terminus, but may contain two or more. It has been found by
the inventors herein that the effects of anthraquinone moieties on
hybridization strength are additive with respect to DNA
hybridization, unlike those of comparable conjugates containing
other substituents such as acridine derivatized to the
oligonucleotide. The strengthened hybridization conferred by the
anthraquinone makes these derivatized oligomers preferred drugs in
antisense therapy, in particular because the specificity of the
oligomer is not lost by virtue of this coupling.
Utility and Administration
Accordingly, the modified oligomers of the invention are useful in
therapeutic, diagnostic and research contexts. In therapeutic
applications, the oligomers are utilized in a manner appropriate
for antisense therapy in general--as described above, antisense
therapy as used herein includes targeting a specific DNA or RNA
sequence through complementarity or through any other specific
binding means, for example, sequence-specific orientation in the
major groove of the DNA double-helix, or any other specific binding
mode. For such therapy, the oligomers of the invention can be
formulated for a variety of modes of administration, including
systemic and topical or localized administration. Techniques and
formulations generally may be found in Remington's Pharmaceutical
Sciences, Mack Publishing Co., Easton, Pa., latest edition.
For systemic administration, injection is preferred, including
intramuscular, intravenous, intraperitoneal, and subcutaneous. For
injection, the oligomers of the invention are formulated in liquid
solutions, preferably in physiologically compatible buffers such as
Hank's solution or Ringer's solution. In addition, the oligomers
may be formulated in solid form and redissolved or suspended
immediately prior to use. Lyophilized forms are also included.
Systemic administration can also be by transmucosal or transdermal
means, or the compounds can be administered orally. For
transmucosal or transdermal administration, penetrants appropriate
to the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art, and include, for
example, for transmucosal administration bile salts and fusidic
acid derivatives. In addition, detergents may be used to facilitate
permeation. Transmucosal administration may be through nasal
sprays, for example, or using suppositories. For oral
administration, the oligomers are formulated into conventional oral
administration forms such as capsules, tablets, and tonics.
For topical administration, the oligomers of the invention are
formulated into ointments, salves, gels, or creams, as is generally
known in the art.
In addition to use in therapy, the oligomers of the invention may
be used as diagnostic reagents to detect the presence or absence of
the target DNA or RNA sequences to which they specifically bind.
Such diagnostic tests are conducted by hybridization through base
complementarity or triple helix formation which is then detected by
conventional means. For example, the oligomers may be labeled using
radioactive, fluorescent, or chromogenic labels and the presence of
label bound to solid support detected. Alternatively, the presence
of a double or triple helix may be detected by antibodies which
specifically recognize these forms. Means for conducting assays
using such oligomers as probes are generally known.
In addition to the foregoing uses, the ability of the oligomers to
inhibit gene expression can be verified in in vitro systems by
measuring the levels of expression in recombinant systems.
It may be commented that the mechanism by which the
specifically-binding oligomers of the invention interfere with or
inhibit the activity of a target RNA or DNA is not always
established, and is not a part of the invention. If the oligomer
seeks, for example, a target mRNA, translation may be inhibited. In
addition, by binding the target, the degradation of the mRNA
message may be enhanced, or the further processing of the RNA may
be inhibited. By formation of a triple helix, the transcription or
replication of the subject DNA may be inhibited; furthermore,
reverse transcription of infectious RNA or replication of
infectious DNA is interfered with. It is also thought that the
immune function may be modulated through physiological mechanisms
similar to those induced by double-stranded RNA as exemplified by
the "ampligen" system or similar to those used to suppress systemic
lupus erythematosus. The oligomers of the invention are
characterized by their ability to target specific oligonucleotide
sequences regardless of the mechanisms of targeting or the
mechanism of the effect thereof.
The following examples are intended to illustrate, but not to
limit, the invention.
EXAMPLE 1
Preparation of Protected Pseudonucleoside Conjugated to
Anthraquinone
A. 2-(N,N-diethanolamino)anthraquinone was prepared as follows. A
mixture of 2-chloroanthraquinone (2.42 g; 10 nmole) and an excess
of diethanolamine in DMSO (20 ml) was heated to 150.degree. C.
After 24 hours reaction, the reaction mixture was cooled to room
temperature, then poured into water (70 ml). The red precipitate
was filtered off, washed thoroughly with water, and dried in air.
The crude product, containing some starting material, was used for
the protection reaction of paragraph B without further
purification.
B. The crude compound prepared in paragraph A, was dissolved in
pyridine (20 ml) and triethyl amine (1.7 ml), cooled to 0.degree.
C., followed by addition of DMAP (0.2 g) and DMT-Cl (4.0 g; 12
mmol). The reaction mixture was warmed to room temperature. After 4
hours of reaction, more DMT-Cl (1 g) was added to the reaction
mixture and reacted one more hour, then concentrated to dryness.
The residue was then partitioned between methylene chloride and
saturated sodium bicarbonate solution. The organic solution was
separated and dried, purified by flash column chromatography on
silica gel, eluted with 1% Et.sub.3 N/1% CH.sub.3 OH/CH.sub.2
Cl.sub.2, to afford the product mono-DMT-protected
2-N,N-diethanolaminoanthraquinone, in an amount of 0.7 g (13%
overall yield); 3.1 g of unreacted starting material was
recovered.
EXAMPLE 2
Conjugation to CPG Support
A. A mixture of the DMT-protected anthraquinone pseudonucleoside of
Example 1, paragraph B (0.5 g; 0.81 mmol), DMAP (0.1 g) and
succinic anhydride (0.326 g; 3.26 mmol) in pyridine (10 ml) was
stirred at room temperature for 4 hours, and more succinic
anhydride (0.1 g) was added to the reaction. After 2 more hours of
reaction, the residue was dissolved in methylene chloride, washed
with 1M TEAB aqueous solution. The organic solution was isolated,
dried over Na.sub.2 SO.sub.4, concentrated, then purified by flash
column chromatography, and eluted with 1% Et.sub.3 N/2% CH.sub.3
OH/CH.sub.2 Cl.sub.2, 1% Et.sub.3 N/5% CH.sub.3 OH/CH.sub.2
CH.sub.2, to afford the succinylated product. The succinylated form
of the subject compound (0.39 g) was isolated as a red solid (yield
59%).
B. After succinylation as described in paragraph A, 3-5 equivalents
of the succinylated pseudonucleoside, 10 equivalents of
diisopropylcarbodiimide, a catalytic amount of DMAP and CPG in
DMF/pyridine (4/1; 4 ml/g CPG) were shaken at room temperature
overnight and then capped with acetic anhydride and pyridine. After
4 hours capping at room temperature, quenching by slow addition of
methanol, CPG was filtered off and washed thoroughly with methylene
chloride, methanol, and ether, and dried under vacuum overnight.
The resulting CPG derivative with anthraquinone-coupled
pseudonucleoside are then used to provide the pseudonucleotide.
EXAMPLE 3
Phosphorylation of Mono-DMT-Protected Pseudonucleosides
To a cold methylene chloride solution (8 ml; 0.degree. C.) of the
DMT-protected anthraquinone pseudonucleoside of Example 1 paragraph
B (0.24 g; 0.39 mmol) were added pyridine (0.1 ml) and
2-chloro-4H-1,2,3-benzodioxaphos-phorin-4-one (1.17 ml of 1M
methylene chloride solution; 1.17 mmol). After 0.5 hours of
reaction at 0.degree. C., the reaction mixture was washed with 1M
TEAB aqueous solution. The organic solution was separated, dried
over Na.sub.2 SO.sub.4, and concentrated. The residue was purified
by flash column chromatography, eluted with 1.5% Et.sub.3 N/5%
CH.sub.3 OH/CH.sub.2 Cl.sub.2. Fractions of the product were
combined, washed with 1M TEAB aqueous solution, dried over Na.sub.2
SO.sub.4, and concentrated, affording phosphorylated DMT-protected
anthraquinone pseudonucleotide.
EXAMPLE 4
Synthesis of Oligomers
The protected anthraquinone-coupled pseudonucleosides of Examples 2
and 3 were then utilized in standard solid-phase oligomer synthesis
techniques, as described in Oligonucleotide Synthesis--A Practical
Approach, Gait, M. J., ed. (1984) IRL Press, Ltd.
EXAMPLE 5
Properties of Oligomers Coupled to Anthraquinone
The oligomers shown below were synthesized and tested for stability
in vitro and in vivo, and specificity of hybridization to
complementary DNA and RNA. The oligomers prepared are as follows,
where "P" represents the pseudonucleotide anthraquinone-containing
residue.
TABLE 1
__________________________________________________________________________
Oligomer .DELTA.T, .degree.C. Tm, .degree.C. No. Oligomer DNA.sup.a
RNA.sup.b RNA.sup.b DNA.sup.a Control
__________________________________________________________________________
1 5'-CCC-TCT-CTT-TTT-CCP +4.5 +4.0 64.0 56.5 (52) 2
5'-CCC-TCT-PCT-TTT-TCC +3.0 -0.5 59.5 55.0 (52) 3
5'-CCC-TCT-CPT-TTT-TCC +2.5 -0.5 59.5 54.5 (52) 4
5'-PCC-CTC-TCT-TTT-TCC +5.0 +4.0 64.0 59.0 (52) 5
5'-CCC-TCT-PCT-TTT-TCC-P +7.5 +4.0 64.0 59.5 (52) 6
5'-PCC-CTC-TCT-TTT-TCC-P +9.0 +8.0 68.0 61.0 (51.5) 7
5'-PCC-CTC-TPC-TTT-TTC-CP +11.0 +7.5 67.5 63.0 (51.5) Control
5'-CCC-TCT-CTT-TTT-CC 0 0 60.0 52.0
__________________________________________________________________________
.sup.a .about.5 .mu.M DNA/DNA (1/1) in 150 mM NaCl/10 mM Na.sub.2
HPO.sub.4, pH 7.5 .sup.b .about.1.6 .mu.M DNA/RNA (1/1) in 150 mM
NaCl/50 mM Tris, pH 7.5
Table 1 above gives the results with respect to stability of
hybridization to complement as a difference in melting point, as
compared to the control lacking the inclusion of the
pseudonucleotide of the invention. As seen from the table,
hybridization is increased with respect to DNA and RNA complement
in almost all cases. It is seen that the oligomers which contain
two anthraquinone modifications, generally, show cumulatively
enhanced stability as compared to those containing only one such
residue.
In a manner similar to that set forth in Examples 1-4 above,
oligomers were constructed which include as "P*", the
pseudonucleoside HO(CH.sub.2).sub.6 NH(CH.sub.2).sub.6 OH wherein
the nitrogen is substituted with anthraquinone at the beta
position. These analogs were tested for stability of hybridization
with complementary RNA. The results, shown in Table 2, indicate the
longer methylene chains in the pseudonucleoside do not result in
enhanced stability as compared to the diethanolamine
pseudobases.
TABLE 2 ______________________________________ .DELTA.T, Oligomer
Tm, .degree.C. .degree.C. ______________________________________ 1*
5'-CCC-TCT-CTT-TTT-CCP* 64.0 4.0 2* 5'-CCC-TCT-P*CT-TTT-TCC 57.5
-2.5 4* 5'-P*CC-CTC-TCT-TTT-TCC 63.5 3.5 5*
5'-CCC-TCT-P*CT-TTT-TCC-P* 58.5 -1.5 6* 5'-P*CC-CTC-TCT-TTT-TCC-P*
65.0 5.0 Control 5'-CCC-TCT-CTT-TTT-CC 60.0 0
______________________________________
Stability to Nuclease Degradation
The stability of the modified oligonucleotides to lysis was tested
under conditions which simulate in vitro cell culture and typical
in vivo environments. The oligomer, 5'-TTT-TTC-TCC-ATP, wherein P
represents diethanolamine pseudonucleoside derivatized to
anthraquinone was prepared, as described in Examples 1-4. The
control is of the formula 5'-TTT-TTC-TCC-AT.
These oligonucleotides were treated with snake venom
phosphodiesterase, under conditions where the control is completely
degraded within 5 minutes. Approximately 90% of the modified
oligomer remained intact at 5 minutes. Table 3 shows the
comparative stability of the control and modified oligomer in
RPMI+10% heat-inactivated fetal calf serum; the data are given as %
oligomer remaining at the noted times. It is clear that
derivatization to anthraquinone enhances stability.
TABLE 3 ______________________________________ 0 hr 1 hr 3 hr 6 hr
______________________________________ Control 100% 0% 0% 0%
Anthraquinone 100% 90% 60% 50%
______________________________________
Specificity of Hybridization
The oligomers containing anthraquinone-conjugated pseudonucleotides
were evaluated with regard to specificity in comparison to
controls. Hybrid oligomer/single-strand RNA complexes were
examined. As shown in Table 4 below, a single basepair mismatch has
a comparable effect on the melting temperature of the oligomer
whether or not the oligomer is coupled to anthraquinone. In the
first set of controls, a single basepair mismatch in the middle of
the sequence lowers the melting temperature by about 8.degree. C.
in the controls; a similar melting point lowering is achieved in
the presence of two anthraquinone residues.
Similarly, when the mismatch is at the 3' end, the presence of the
anthraquinone residue enhances the discrimination of a mismatch
thereby improving the specificity. However, the anthraquinone
moiety enhances the discrimination of a mismatch, thereby improving
the specificity of hybridization.
TABLE 4 ______________________________________ Tm, .degree.C.
.DELTA.T, .degree.C. ______________________________________ Effect
of Mismatch on Controls: 5'-CCC-TCT-CTT-TTT-CC 60.5 --
5'-T-CCC-TCT-CTT-TTT-CC 61.0 +0.5 5'-CCC-TCT-TTT-TTT-CC 52.5 -8.0
Effect of Mismatch with Anthraquinone: 5'-P-CCC-TCT-CTT-TTT-CCP
68.0 -- 5'-P-CCC-TCT-TTT-TTT-CCP 60.5 -7.5 Effect of 3'-Terminal
Mismatch on Control: 5'-CCC-TCT-CTT-TTT-CC 60.0 --
5'-CCC-TCT-CTT-TTT-CT 57.5 -2.5 Effect of 3'-Terminal Mismatch with
Anthraquinone: 5'-CCC-TCT-CTT-TTT-CC-P 64 --
5'-CCC-TCT-CTT-TTT-CT-P 59.5 -4.5
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